Trypanosoma brucei is a group of hemoflagellated parasitic protozoa that causes a fatal disease in humans known as African sleeping sickness and a similar diseases in domestic animals called nagana. The disease is transmitted by an insect vector, the tsetse fly. The procyclic form of the parasite, found inside the insect's alimentary gut, possesses a fully developed mitochondrion. On the other hand, the bloodstream form of the parasite that grows in the mammalian blood and tissue fluids possesses a developmentally suppressed mitochondrion having no cytochromes. Trypanosome Alternative Oxidase (TAO) is a nuclear encoded protein, which serves as the only terminal oxidase in the mitochondria of the bloodstream form of T.brucei. TAO protein needs to be properly imported into mitochondria to perform its function. TAO possesses a predicted N-terminal mitochondrial targeting sequence. In fungi and higher eukaryotes, preproteins containing the N-terminal targeting sequence depend on mitochondrial membrane potential for their import into mitochondria. However, a recent finding in our laboratory has shown that in the bloodstream form of the parasite, inner membrane potential is not required for TAO import whereas in the procyclic form, the same precursor protein depends fully on mitochondrial membrane potential for import. Thus, I hypothesize that different targeting signal and translocator proteins are involved for import of TAO into the mitochondria of the bloodstream form in comparison to that in the procyclic form and also that could be different from the protein import molecules found in the host. I proposed to test this hypothesis by the following two specific aims: 1) to elucidate the mitochondrial targeting signal that is necessary and sufficient for import of TAO into the mitochondria of bloodstream and procyclic forms and 2) to delineate the translocase protein(s) used to import TAO into mitochondria of the procyclic and bloodstream forms. I will address these two specific aims using in vitro and in vivo import assays, protein crosslinking, and proteomics. African trypanosomiasis heavily affects sub-Saharan Africa and parts of India. Current treatments, in addition to being toxic to the mammalian host, have multiple targeting sites or pathways in the parasite which makes it easy for the parasite to develop some form of resistance to these therapeutic agents. Elucidation of the mechanism for TAO import in T.brucei, will enable the identification of unique proteins or pathways of protein import that can be used as very defined targets for chemotherapy to prevent T.brucei infection in humans.